This is a proposal to investigate the cellular mechanisms that regulate the extent of signaling by the Gi- coupled m2 muscarinic acetylcholine receptors (m2Rs). These important regulators of cardiovascular function counteract the effects of adrenaline to either stimulate myopulmonary contraction or to decrease the rate and force of myocardial contraction. Limiting m2R signaling is critical for normal cardiac function since persistent activation (e.g., Sjogren's syndrome) often leads to congestive heart failure. One key mechanism that contributes to limiting m2R activity is receptor endocytosis. Although much is known about the mechanisms leading to m2R internalization (e.g., m2R phosphorylation) very little is known about how the subsequent post-endocytic trafficking of m2Rs is regulated. Members of the ARF and Rab families of GTPases are known to regulate the compartment-to-compartment trafficking of nutrient receptors like those that bind LDL or transferrin. In contrast, nothing is known about the role of these GTPases in regulating the intracellular trafficking and function of any muscarinic receptor class. Our recent observations suggest that the ARF6 GTPase, which regulates a non-clathrin mediated endocytic pathway, may regulate the trafficking of m2Rs, which are also internalized via an ill-defined, non-clathrin dependent mechanism. These preliminary results provide support for the CENTRAL HYPOTHESIS of this proposal that specific ARF (ARF6) and Rab (Rabs 5 and 7) regulate the intracellular trafficking and sorting of internalized m2Rs. TO TEST THIS HYPOTHESIS, we will use a combination of molecular, morphological, and biochemical approaches to focus on the following THREE SPECIFIC AIMS. FIRST, we will define the post-endocytic itinerary of internalized m2Rs in cells. SECOND, we will determine the role of specific ARF(6) and Rab(5,7) GTPases in regulating multiple aspects of m2R trafficking. THIRD, we will investigate the role of these GTPases in regulating m2R-mediated ERK2 activation. The information gathered by this proposal will provide new information about the regulation of post-endocytic m2R trafficking and will lend insight into the relationship between ARF6-regulated and clathrin-dependent endocytic trafficking.